Eukaryotic cell division proceeds through a highly regulated cell cycle comprising consecutive phases termed G1, S, G2 and M. Disruption of the cell cycle or cell cycle control can result in cellular abnormalities or disease states such as cancer which arise from multiple genetic changes that transform growth-limited cells into highly invasive cells that are unresponsive to normal control of growth. Transition of normal cells into cancer cells can arise though loss of correct function in DNA replication and DNA repair mechanisms. All dividing cells are subject to a number of control mechanisms, known as cell-cycle checkpoints, which maintain genomic integrity by arresting or inducing destruction of aberrant cells. Investigation of cell cycle progression and control is consequently of significant interest in designing anticancer drugs (Flatt, P. M. and Pietenpol, J. A. Drug Metab. Rev., (2000), 32(3-4), 283-305; Buolamwini, J. K. Current Pharmaceutical Design, (2000), 6, 379-392).
Cell cycle progression is tightly regulated by defined temporal and spatial expression, localisation and destruction of a number of cell cycle regulators which exhibit highly dynamic behaviour during the cell cycle (Pines, J., Nature Cell Biology, (1999), 1, E73-E79). For example, at specific cell cycle stages some proteins translocate from the nucleus to the cytoplasm, or vice versa, and some are rapidly degraded. For details of known cell cycle control components and interactions, see Kohn, Molecular Biology of the Cell (1999), 10, 2703-2734.
Accurate determination of cell cycle status is a key requirement for investigating cellular processes that affect the cell cycle or are dependent on cell cycle position. Such measurements are particularly vital in drug screening applications where:    i) substances which directly or indirectly modify cell cycle progression are desired, for example, for investigation as potential anti-cancer treatments;    ii) drug candidates are to be checked for unwanted effects on cell cycle progression; and/or    iii) it is suspected that an agent is active or inactive towards cells in a particular phase of the cell cycle.
Traditionally, cell cycle status for cell populations has been determined by flow cytometry using fluorescent dyes which stain the DNA content of cell nuclei (Barlogie, B. et al, Cancer Res., (1983), 43(9), 3982-97). Flow cytometry yields quantitative information on the DNA content of cells and hence allows determination of the relative numbers of cells in the G1, S and G2+M phases of the cell cycle. However, this analysis is a destructive non-dynamic process and requires serial sampling of a population to determine cell cycle status with time. A further disadvantage of flow cytometry techniques relates to the indirect and inferred assignment of cell cycle position of cells based on DNA content. Since the DNA content of cell nuclei varies through the cell cycle in a reasonably predictable fashion, ie. cells in G2 or M have twice the DNA content of cells in G1, and cells undergoing DNA synthesis in S phase have an intermediate amount of DNA, it is possible to monitor the relative distribution of cells between different phases of the cell cycle. However, the technique does not allow precision in determining the cell cycle position of any individual cell due to ambiguity in assigning cells to G2 or M phases and to further imprecision arising from inherent variation in DNA content from cell to cell within a population which can preclude precise discrimination between cells which are close to the boundary between adjacent phases of the cell cycle. Additionally, variations in DNA content and DNA staining between different cell types from different tissues or organisms require that the technique is optimised for each cell type, and can complicate direct comparisons of data between cell types or between experiments (Herman, Cancer (1992), 69(6), 1553-1556). Flow cytometry is therefore suitable for examining the overall cell cycle distribution of cells within a population, but cannot be used to monitor the precise cell cycle status of an individual cell over time.
EP 798386 describes a method for the analysis of the cell cycle of cell sub-populations present in heterogeneous cell samples. This method uses sequential incubation of the sample with fluorescently labelled monoclonal antibodies to identify specific cell types and a fluorochrome that specifically binds to nucleic acids. This permits determination of the cell cycle distribution of sub-populations of cells present in the sample. However, as this method utilises flow cytometry, it yields only non-dynamic data and requires serial measurements to be performed on separate samples of cells to determine variations in the cell cycle status of a cell population with time following exposure to an agent under investigation for effects on cell cycle progression.
A number of researchers have studied the cell cycle using traditional reporter enzymes that require the cells to be fixed or lysed. For example Hauser & Bauer (Plant and Soil, (2000), 226, 1-10) used β-glucuronidase (GUS) to study cell division in a plant meristem and Brandeis & Hunt (EMBO J., (1996), 15, 5280-5289) used chloramphenical acetyl transferase (CAT) fusion proteins to study variations in cyclin levels. U.S. Pat. No. 6,048,693 describes a method for screening for compounds affecting cell cycle regulatory proteins, wherein expression of a reporter gene is linked to control elements which are acted on by cyclins or other cell cycle control proteins. In this method, temporal expression of a reporter gene product is driven in a cell cycle specific fashion and compounds acting on one or more cell cycle control components may increase or decrease expression levels.
U.S. Pat. No. 6,159,691 describes nuclear localisation signals (NLS) derived from the cell cycle phase-specific transcription factors DP-3 and E2F-1 and claims a method for assaying for putative regulators of cell cycle progression. In this method, nuclear localisation signals (NLS) derived from the cell cycle phase specific transcription factors DP-3 and E2F-1 may be used to assay the activity of compounds which act to increase or decrease nuclear localisation of specific NLS sequences from DP-3 and E2F-1 fused to a detectable marker.
Jones et al (Nat Biotech., (2004), 23, 306-312) describe a fluorescent biosensor of mitosis based on a plasma membrane targeting signal and an SV40 large T antigen NLS fused to EYFP. Throughout the cell cycle the reporter resides in the nucleus but translocates to the plasma membrane during mitosis, between nuclear envelope breakdown and re-formation.
WO 03/031612 describes DNA reporter constructs and methods for determining the cell cycle position of living mammalian cells by means of cell cycle phase-specific expression control elements and destruction control elements.
Gu et al. (Mol Biol Cell., 2004,15, 3320-3332) have recently investigated the function of human DNA helicase B (HDHB) and shown that it is primarily nuclear in G1 and cytoplasmic in S and G2 phases, that it resides in nuclear foci induced by DNA damage, that the focal pattern requires HDHB activity, and that HDHB localization is regulated by CDK phosphorylation.
None of the preceding methods specifically describe sensors which can be stably integrated into the genome and used to indicate G1, S and G2 phases of the cell cycle. Consequently, methods are required that enable these phases of the cell cycle to be determined non-destructively in a single living mammalian cell, allowing the same cell to be repeatedly interrogated over time, and which enable the study of the effects of agents having potentially desired or undesired effects on the cell cycle. Methods are also required that permit the parallel assessment of these effects for a plurality of agents.